Screen for sodium channel modulators

ABSTRACT

A method or screen for assessing the potential of a compound to treat a pathological condition, such as arrhythmia, which is manifested by an increased late sodium current in a heart is disclosed. The method employs a mutant sodium channel protein having an amino acid sequence in which one or more amino acids among the ten amino acids occurring at the carboxy end of the S6 segments of D1, D2, D3 or D4 domains of mammalian Nav1 differs from the amino acid in wild-type Nav1 by substitution with tryptophan, phenylalanine, tyrosine or cysteine. Cells transfected with a nucleic acid that encodes a mutant mammalian Nav1 protein, as well as isolated nucleic acids comprising a nucleotide sequence that codes for a mutant mammalian Nav1 protein are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of copending U.S. Ser. No.10/608,584 filed Jun. 26, 2003, the disclosure of which is herebyincorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made under grant number 5RO1HL6607602 from theNational Heart, Lung and Blood Institute. The government may havecertain rights in the invention.

FIELD OF THE INVENTION

The invention relates to a method for screening compounds for use asanti-arrhythmic agents. The method employs a cell line that expresses amutant sodium channel protein.

BACKGROUND OF THE INVENTION

Mammalian voltage-gated sodium channels are pore-forming membraneproteins responsible for the initiation and propagation of actionpotentials in excitable membranes in nerve, skeletal muscle and heartcells. The controlled gating of sodium channels in response to membranedepolarizations is necessary for normal electrical signaling andestablishing of intercellular communication. Voltage-gated Na+ ionchannels consist of one large α-subunit (about 200 kDa) and one or twosmaller β-subunits. The α-subunits are designated “Nav” (Na for sodiumchannel and v for voltage-gated), followed by a numbering system for theparticular isoform. The Na+ channel α-subunit isoforms contain fourhomologous repeated domains (D1-D4) each with six transmembrane segments(S1-S6). The α-subunit protein alone forms a functional channel whenexpressed in mammalian expression systems. The four repeated domains arehypothesized to assemble as a peudotetrameric structure with thepermeation pathway situated at the center. FIG. 1 is a cartoon depictingone conceptualization of how the Nav protein arranges itself withrespect to the membrane. The cartoon is not accurate; it is an expandedmodel that does not attempt to depict how the four S6 segments cometogether to form the sodium channel, but it facilitates an understandingof how the proteins might align with respect to the inside and outsideof the excitable membrane. In fact, recent studies suggest that four S6C-termini may jointly close the voltage-gated cation channel at thecytoplasmic side, probably as an inverted teepee structure.

Several pieces of evidence suggest that S6 segments are involved in Na+channel gating. First, a number of receptors for various therapeuticdrugs and neurotoxins such as local anesthetics (LAs), antiarrhythmics,anticonvulsants, antidepressants, pyrethroid insecticides, batrachotoxin(BTX), and veratridine, are situated at the middle of multiple S6segments. Upon binding, these ligands exert their pharmacologicalactions on the Na+ channel, presumably in part via their correspondingS6 receptor. In particular, BTX drastically modifies Na+ channelactivation, fast inactivation, and slow inactivation, suggesting thatits receptor is linked to these gating processes.

The invention herein described arose from a hypothesis that S6 segmentsmay be structurally geared for channel activation by lateral/rotationalmovement via a flexible gating hinge, a glycine or serine residuelocated at the middle of the inner Na+ channel S6 segments. This gatinghinge could have two different conformations. One is in its relaxedstraight α-helical form, which closes the channel at the S6 C-terminalend, and the other is the bendable α-helical form, which may bendoutward at a 30° angle and thus splay open the channel at the S6constricted C-terminus. After channel activation, S6 segments may thenform the docking site for the fast-inactivation gate. A putative Na+channel inactivation gate has been delineated at the intracellularlinker between D3 and D4 by West et al. [Proc. Natl. Acad. Sci. USA89:10910-10914 (1992)]. This linker could be situated at the C-terminiof S6 segments, where the inactivation gate may plug the open channelwhile it binds to its docking site. This plugging mechanism has recentlybeen demonstrated in voltage-gated K+ channels [Zhou et al., Nature411:657-661 (2001)]. The foregoing hypothesis is useful because itprovides a framework for interpreting the results and makingpredictions. However, it is important to note that the invention isbased on the results, not the hypothesis, and the hypothesis should notbe viewed as a limitation on the claimed invention.

There is very close homology among the S6 segments of mammalian Navproteins so far identified. This homology extends both through speciesand through isoforms of the Nav protein. As can be seen in thecomparison below, the few variations that exist among the amino acids inthe amino terminal portion of the S6 segments are very conservativereplacements, and the carboxy terminal 11 amino acids of the S6 segmentsof all four domains are identical for rats and humans for both of themuscle sodium channel proteins Nav1.4 and Nav1.5: D1S6       1     6     11    16    21    26 human Nav1.1 YMIFF VLVIF LGSFYLINLI LAVVA MAY (SEQ ID NO.: 1) Nav1.2 YMIFF VLVIF LGSFY LINLI LAVVA MAY(SEQ ID NO.: 2) Nav1.3 YMIFF VLVIF LGSFY LINLI LAVVA MAY (SEQ ID NO.: 3)Nav1.4 YMIFF VVIIF LGSFY LINLI LAVVA MAY (SEQ ID NO.: 4) Nav1.5 YMIFFMLVIF LGSFY LVNLI LAVVA MAY (SEQ ID NO.: 5) Nav1.8 YMIFF vVvIF LGSFYLVNLI LAVVA MAY (SEQ ID NO.: 6) Nav1.9 YMIFF VVVIF LGSFY LINLI LAVVA MAY(SEQ ID NO.: 7) rat Nav1.4 YMIFF VVIIF LGSFY LINLI LAVVA MAY (SEQ IDNO.: 8) Nav1.5 YMIFF MLVIF LGSFY LVNLI LAVVA MAY (SEQ ID NO.: 9) Nav1.6YMIFF MLVIF VGSFY PVNLI LAVVA MAY (SEQ ID NO.: 10) Nav1.7 YMVFF VVVIFLGSFY LVNLI LAVVA MAY (SEQ ID NO.: 11) Nav1.8 YMVFF MLVIF LGSFY LVNLILAVVA MAY (SEQ ID NO.: 12) D2S6        1     6     11    16    21    26human Nav1.1 CLTVF MMVMV IGNLV VLNLF LALLL SSF (SEQ ID NO.: 13) Nav1.2CLTVF MMVMV IGNLV VLNLF LALLL SSF (SEQ ID NO.: 14) Nav1.3 CLIVF MLVMVIGNLV VLNLF LALLL SSF (SEQ ID NO.: 15) Nav1.5 CLLVF LLVMV IGNLV VLNLFLALLL SSF (SEQ ID NO.: 16) rat Nav1.4 CLTVF LMVMV IGNLV VLNLF LALLL SSF(SEQ ID NO.: 17) Nav1.5 CLLVF LLVMV IGNLV VLNLF LALLL SSF (SEQ ID NO.:18) D3S6        1     6     11    16    21    26 human Nav1.1 MYLYFVIFII FGSFF TLNLF IGVII DNF (SEQ ID NO.: 19) Nav1.2 MYLYF VIFII FGSFFTLNLF IGVII DNF (SEQ ID NO.: 20) Nav1.3 MYLYF VIFII FGSFF TLNLF IGVIIDNF (SEQ ID NO.: 21) Nav1.4 MYLYF VIFII FGSFF TLNLF IGVII DNF (SEQ IDNO.: 22) Nav1.5 MYIYF VIFII FGSFF TLNLF IGVII DNF (SEQ ID NO.: 23)Nav1.8 MYLYF VIFII GGSFF TLNLF VGVII DNF (SEQ ID NO.: 24) rat Nav1.4MYLYF VIFII FGSFF TLNLF IGVII DNF (SEQ ID NO.: 25) Nav1.5 MYIYF VVFIIFGSFF TLNLF IGVII DNF (SEQ ID NO.: 26) Nav1.7 MYLYF VVFII FGSFF TLNLFIGVII DNF (SEQ ID NO.: 27) Nav1.8 MYIYF VVFII FGGFF TLNLF VGVII DNF (SEQID NO.: 28) D4S6        1     6     11    16    21    26 human Nav1.1GIFFF VSYII ISFLV VVNMY IAVIL ENF (SEQ ID NO.: 29) Nav1.2 GIFFF VSYIIISFLV VVNMY IAVIL ENF (SEQ ID NO.: 30) Nav1.3 GIFFF VSYII ISFLV VVNMYIAVIL ENF (SEQ ID NO.: 31) Nav1.4 GICFF CSYII ISFLI VVNMY IAIIL ENF (SEQID NO.: 32) Nav1.5 GILFF TTYII ISFLI VVNMY IAIIL ENF (SEQ ID NO.: 33)rat Nav1.4 GICFF CSYII ISFLI VVNMY IAIIL ENF (SEQ ID NO.: 34) Nav1.5GILFF TTYII ISFLI VVNMY IAIIL ENF (SEQ ID NO.: 35)

Except for a single I→V change at position 7 of D3S6, the rat and humanNav1.4 and Nav1.5 sequences are identical for all four S6 segments.Because of the very high degree of conservation (in fact identity) ofthe 11 amino acids at the carboxy termini of the S6 segments, the personof skill in the art expects that substitution in this region will havethe same effect on sodium channel function across mammalian species andacross isoforms of the Nav1 protein.

The numbering shown in the charts above is the standard numbering usedto identify the 28 amino acids in the S6 segments by their positionwithin that segment. A separate system of numbering that may be appliedto those same amino acids derives from their position within thesequence of the whole protein. Because the amino acid sequences ofmembers of the Nav family of proteins vary widely outside thetransmembrane regions, the protein sequence residue numbers assigned tothe corresponding amino acids in the S6 segments differs among speciesand among sodium channel protein isoforms within species. Thus, theleucine identified as residue 19 in segment 6 in domain 1 (D1S6) is L407in human Nav1.5, L408 in rat Nav1.5, L441 in human Nav1.4 and L435 inrat Nav1.4. Similarly, the isoleucine identified as residue 23 insegment 6 in domain 4 (D4S6) is I1770 in human Nav1.5, I11771 in ratNav1.5, I1581 in human Nav1.4 and I1589 in rat Nav1.4. Unless otherwisenoted, amino acids will be identified hereinafter, when referring to thewhole protein, according to their position in rNav1.4. Thus A438 refersto the alanine that occurs at position 438 in rNav1.4. The invention,however, is not intended to be limited to polypeptides having sequencesderived from the rat; rather, corresponding mammalian sequences,including human are encompassed by the invention.

SUMMARY OF THE INVENTION

In one aspect the invention relates to a method or screen for assessingthe potential of a compound to treat a pathological condition, such asarrhythmia, which is manifested by an increased late sodium current in aheart. The method comprises:

-   (a) providing a recombinant cell that expresses a mutant Nav 1    sodium channel protein;-   (b) measuring a first plateau current in the cell;-   (c) exposing the cell to a test compound;-   (d) measuring a second plateau current in the cell; and-   (e) comparing the first and second currents. A lower second current    indicates that the test compound is a potential anti-arrhythmic    agent. The mutant sodium channel protein has an amino acid sequence    in which one or more amino acids among the ten amino acids occurring    at the carboxy end of the S6 segments of D1, D2, D3 or D4 domains of    mammalian Nav1 differs from the amino acid in wild-type Nav1 by    substitution with tryptophan, phenylalanine, tyrosine or cysteine.    Mammalian Nav 1 proteins encompassed by the present invention    encompass mammalian Nav1.1-Nav 1.9.

In another embodiment, the mutant sodium channel protein has an aminoacid sequence in which one or more amino acids among the ten amino acidsoccurring at the carboxy end of the S6 segments of D1, D2, D3 or D4domains of mammalian Nav1.4 or Nav 1.5 differs from the amino acid inwild-type Nav1 by substitution with tryptophan, phenylalanine, tyrosineor cysteine.

In another embodiment, the mutant sodium channel protein has an aminoacid sequence in which at least one of amino acids 19, 21 and 22 of theS6 segment of D1 and amino acids 23 and 24 of the S6 segment of the D4domain of mammalian Nav1 differs from the amino acid in wild-type Nav1by substitution with tryptophan, phenylalanine, tyrosine or cysteine.

In another embodiment, the mutant sodium channel protein has an aminoacid sequence in which at least one of amino acids 19, 21 and 22 of theS6 segment of D1 and amino acids 23 and 24 of the S6 segment of the D4domain of mammalian Nav1.4 or Nav1.5 differs from the amino acid inwild-type Nav1.4 or Nav1.5 by substitution with tryptophan,phenylalanine, tyrosine or cysteine.

In another embodiment, the mutant sodium channel protein has an aminoacid sequence in which at least one of amino acids L435, L437, A438,I1589 and I1590 of wild-type rNav1.4 is replaced by tryptophan,phenylalanine or tyrosine. L437 of rNav1.4 may be replaced by cysteine,in addition to tryptophan, phenylalanine or tyrosine.

In another aspect, the invention relates to an isolated nucleic acidcomprising a nucleotide sequence that codes for a mutant mammalian Nav 1protein. The mutant protein has a sequence as described above.

In another aspect, the invention relates to a cell transfected with anucleic acid that encodes a mutant mammalian Nav1 protein. The mutantprotein has a sequence as described above.

In another aspect, the invention relates to a functional recombinantsodium channel protein containing an amino acid sequence chosen from:WILAVVAMAY SEQ ID NO.: 36 YILAVVAMAY SEQ ID NO.: 37 FILAVVAMAY SEQ IDNO.: 38 LILWVVAMAY SEQ ID NO.: 39 LILYVVAMAY SEQ ID NO.: 40 LILFVVAMAYSEQ ID NO.: 41 LICWVVAMAY SEQ ID NO.: 42 LICYVVAMAY SEQ ID NO.: 43LICFVVAMAY SEQ ID NO.: 44 WICWVVAMAY SEQ ID NO.: 45 YICYVVAMAY SEQ IDNO.: 46 FICFVVAMAY SEQ ID NO.: 47 WICYVVAMAY SEQ ID NO.: 48 WICFVVAMAYSEQ ID NO.: 49 YICWVVAMAY SEQ ID NO.: 50 FICWVVAMAY SEQ ID NO.: 51YICYVVAMAY SEQ ID NO.: 52 FICFVVAMAY SEQ ID NO.: 53 YICFVVAMAY SEQ IDNO.: 54 FICYVVAMAY SEQ ID NO.: 55 LIWAVWAMAY SEQ ID NO.: 56 LIYAVWAMAYSEQ ID NO.: 57 LIFAVWAMAY SEQ ID NO.: 58 LILAVWAMAY SEQ ID NO.: 59MYIAWILENF SEQ ID NO.: 60 MYIAYILENF SEQ ID NO.: 61 MYIAFILENF SEQ IDNO.: 62 MYIAIWLENF SEQ ID NO.: 63 MYIAIYLENF SEQ ID NO.: 64 MYIAIFLENFSEQ ID NO.: 65 MYIACILENF SEQ ID NO.: 66 MYIAICLENF SEQ ID NO.: 67MYIAWWLENF SEQ ID NO.: 68 MYIAYYLENF SEQ ID NO.: 69 MYIAFFLENF SEQ IDNO.: 70

In another aspect, the invention relates to a functional recombinantsodium channel protein containing two sequences of amino acids. Thefirst amino acid sequence is chosen from: WILAVVAMAY (SEQ ID NO.: 36);LILWVVAMAY (SEQ ID NO.: 39); LICWVVAMAY (SEQ ID NO.: 42); WICWVVAMAY(SEQ ID NO.: 45), and LILAVWAMAY (SEQ ID NO.: 59). The second amino acidsequence chosen from: MYIAWILENF (SEQ ID NO.: 60); MYIAIWLENF (SEQ IDNO.: 63); MYIACILENF (SEQ ID NO.: 66); MYIAICLENF (SEQ ID NO.: 67), andMYIAWWLENF (SEQ ID NO.: 68).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a Nav 1. Na⁺ channel protein ina cell membrane.

FIG. 2A-C illustrates the activation of wild-type Nav 1.4 (A) and rNav1.4-A438W (B) co-expressed with β1. In 2C normalized membraneconductance (g_(m)) is plotted against pulse voltage for wild type (∘)and rNav 1.4-A438W (●)

FIG. 3 is a graphic summary of the effects of W- and selectedC-mutations at C-termini of D1S6 and D4S6 on activation and inactivationgating.

FIG. 4 is a bar graph depicting the relative maintained currents invarious W- and C-mutations at C-termini of D1S6 and D4S6. Fraction ofnon-inactivating current for D1S6 mutants (left), D4S6 (middle), anddouble and triple mutants (right).

FIG. 5A-C shows the steady-state inactivation of wild-type (A) andrNav1.4-A438W (B) coexpressed with β1. Normalized Na⁺ currentavailability (h_(∞)) of wild-type (∘) and rNav1.4-A438W (●) were plottedas a function of the 100-ms conditioning pulse voltage (C).

FIG. 6A-C shows gating properties of rNav1.4-I1589W coexpressed with β1.

FIG. 7A-C shows activation of rNav1.4-A438C (A) and rNav1.4-I1589C (B)coexpressed with β1. Normalized membrane conductance (g_(m)) is polttedagainst the amplitude of the 5-ms voltage step in (C).

FIG. 8A-D shows activation gating of double and triple mutantscotransfected with β1: A438W/I1589W (A); L437C/A438W (B);L435W/L437C/A438W (C); I1589W/I1590W (D).

FIG. 9A-B shows slow inactivation gating of double and triple mutants.Normalized peak current is plotted against conditioning pre-pulsepotential for each (A). The development of slow inactivation (B).

FIG. 10 shows a coarse correlation between fast and slow inactivationgating. (A) The relative level of slow inactivation for D1S6 mutants(left), D4S6 (middle), and double and triple mutants (right). (B) Thefraction of slow-inactivated current versus the fraction ofnoninactivating current of the individual mutant.

FIG. 11 depicts voltage dependence of flecainide block in rNav1.4channels.

FIG. 12 shows blockade of inactivation-deficient Nav1.4L435W/L437C/A438W channels at various flecainide concentrations.Superimposed Na⁺ currents evoked by a 140 ms test pulse to +30 mV every30 seconds were shown at various flecainide concentrations. Steady stateblock at each concentration was achieved within 5 min.

FIG. 13 shows the decay phase of the Na⁺ current.

FIG. 14A-D shows gating properties of hNav1.4-L443C/A444W mutantchannels in stably transfected HEK293 cells. (A) Superimposed Na⁺current traces evoked by 8-ms pulses to voltages ranging from −100 to+50 mV in 10-mV increments. The inward current evoked by a pulse to −40mV and the outward current evoked by a pulse to +50 mV are labeled. (B)Conductance plotted against the corresponding voltage. Superimposedcurrent traces evoked by an 8-ms test pulse to +50 mV with 100-msconditioning pulses, increased in 5-mV increments between −170 and −25mV. The interval between pulses was 10 seconds. (D) Normalized Na⁺current availability (h_(∞)) plotted against conditioning voltage.

FIG. 15A-B demonstrates that BTX prevents the slow decay ofhNav1.4-L443C/A444W mutant Na⁺ currents.

FIG. 16 demonstrates that TTX blocks hNav1.4-L443C/A444W Na⁺ channels.(A) Superimposed Na⁺ current traces were recorded in the absence and inthe presence of TTX ranging from 3 nM to 1 μM. Currents were evoked by a3-ms test pulse to +50 mV every 30 seconds. A steady state at eachconcentration was reached before application of the next concentratedsolution. (B) The dose-response curve was constructed using the data setas shown in (A). The peak currents were measured, normalized to theamplitude of the control, and plotted against the TTX concentration.Solid line represents a fit to the data with the Hill equation. Theestimated IC₅₀ value±standard error [Hill coefficient±S.E.] was 15.1±0.6nM [1.09±0.04] (n=5).

FIG. 17 graphically illustrates block of hNav1.4-L443C/A444W Na⁺channels by antiarrhythmic drugs. Superimposed Na⁺ currents wererecorded before and after 10 μM flecainide (A) and mexiletine (B).Currents of hNav1.4-L443C/A444W Na⁺ channels were evoked by a 100-mstest pulse at +50 mV. Drugs were applied externally. The currents wererecorded after the open-channel block reached its steady state, usuallywithin 5 min. A different cell was used in each panel. Holding potentialwas −140 mV.

FIG. 18 graphically illustrates the Block of hNav1.4-L443C/A444W Na⁺channels by propafenone and amiodarone. Superimposed Na⁺ currents wererecorded before and after 10 μM (S)-propafenone enantiomer (A) andamiodarone (B). Currents of hNav1.4-L443C/A444W Na⁺ channels were evokedby a 100-ms test pulse at +50 mV. Drugs were applied externally. Thecurrents were recorded after propafenone block reached its steady state,usually within 5 min (A). In contrast, wash-in for amiodarone at 10 μMwas slow; Na⁺ currents were decreased continuously over a period of 15min (B). A different cell was used in each panel. Holding potential was−140 mV.

DETAILED DESCRIPTION OF THE INVENTION

All patents, applications, publications and other references citedherein are hereby incorporated by reference in their entirety into thepresent application.

The first aspect of the invention relates to a screen for assessing thepotential of a compound to treat a pathological condition, such asarrhythmia, which is manifested by an increased late sodium current in aheart. The method comprises:

-   (a) providing a cell that expresses a recombinant mutant Nav1 sodium    channel protein;-   (b) measuring a first plateau current in the cell;-   (c) exposing the cell to a test compound;-   (d) measuring a second plateau current in the cell; and-   (e) comparing the first and second currents. A lower second current    indicates that the test compound is a potential anti-arrhythmic    agent. The mutant sodium channel protein has an amino acid sequence    in which one or more amino acids among the ten amino acids occurring    at the carboxy end of the S6 segments of D1, D2, D3 or D4 domains of    a mammalian Nav1 differs from the amino acid in wild-type Nav1.4 or    by substitution with tryptophan, phenylalanine, tyrosine or    cysteine. In a preferred embodiment, the mutant sodium channel    protein has an amino acid sequence in which at least one amino acid    chosen from amino acids 19, 21 and 22 of the S6 segment of D1 and    amino acids 23 and 24 of the S6 segment of the D4 domain of a    mammalian Nav1 is the amino acid that is replaced. These amino acids    correspond to amino acids L435, L437, A438, I1589 and I1590 of    wild-type rNav1.4. The wild-type amino acids may be replaced by    tryptophan, phenylalanine or tryrosine, all of which are neutral,    hydrophobic and bulky—the important common features for impairing    the so-called “fast inactivation” of the sodium channel. Certain of    the wild-type amino acids may also be replaced by cysteine. Cysteine    produces a similar impairment of fast inactivation, but appears to    do so by an indirect route, whereby it achieves effective bulkiness    (and hydrophobicity) through reaction of the sulfhydryl with    physiologically accessible nucleophiles. The experiments described    below were carried out with tryptophan and cysteine.

The remainder of the Nav protein—outside the S6 segments—is optimallythe sequence of a Nav 1 sodium channel protein, for example, Nav 1.1,Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, Nav 1.8 or Nav1.9. Nav1.4 and Nav1.5 are the two isoforms of the Nav protein that arefound in skeletal and heart muscle; the remaining isoforms have beenprimarily identified in the CNS and neuronal structures. In oneembodiment, a leucine corresponding to L437 of rNav1.4 is replaced withcysteine, and one or both of a leucine and an alanine corresponding toL435 and A438 respectively are replaced with tryptophan. In otherembodiments, alanine corresponding to A438 and an isoleucinecorresponding to I1589 are replaced, preferably by tryptophan. Preferredsequences of the residues 19-28 of the S6 segment are: WILAVVAMAY SEQ IDNO.: 36 YILAVVAMAY SEQ ID NO.: 37 FILAVVAMAY SEQ ID NO.: 38 LILWVVAMAYSEQ ID NO.: 39 LILYVVAMAY SEQ ID NO.: 40 LILFVVAMAY SEQ ID NO.: 41LICWVVAMAY SEQ ID NO.: 42 LICYVVAMAY SEQ ID NO.: 43 LICFVVAMAY SEQ IDNO.: 44 WICWVVAMAY SEQ ID NO.: 45 YICYVVAMAY SEQ ID NO.: 46 FICFVVAMAYSEQ ID NO.: 47 WICYVVAMAY SEQ ID NO.: 48 WICFVVAMAY SEQ ID NO.: 49YICWVVAMAY SEQ ID NO.: 50 FICWVVAMAY SEQ ID NO.: 51 YICYVVAMAY SEQ IDNO.: 52 FICFVVAMAY SEQ ID NO.: 53 YICFVVAMAY SEQ ID NO.: 54 FICYVVAMAYSEQ ID NO.: 55 LIWAVWAMAY SEQ ID NO.: 56 LIYAVWAMAY SEQ ID NO.: 57LIFAVWAMAY SEQ ID NO.: 58 LILAVWAMAY SEQ ID NO.: 59 MYIAWILENF SEQ IDNO.: 60 MYIAYILENF SEQ ID NO.: 61 MYIAFILENF SEQ ID NO.: 62 MYIAIWLENFSEQ ID NO.: 63 MYIAIYLENF SEQ ID NO.: 64 MYIAIFLENF SEQ ID NO.: 65MYIACILENF SEQ ID NO.: 66 MYIAICLENF SEQ ID NO.: 67 MYIAWWLENF SEQ IDNO.: 68 MYIAYYLENF SEQ ID NO.: 69 MYIAFFLENF SEQ ID NO.: 70

The resultant protein may also contain a second amino acid sequence,YMIFFX^(a)X^(b)X^(c)IFLGSFYLX^(d)N (SEQ ID NO. 71), amino-terminal tothe foregoing amino acid sequence. In the second sequence, X^(a) is V orM; X^(b) is L or V; and X^(c) and X^(d) are independently I or V. Thesevariable residues account for the variants thus far observed in S6residues 1-18 of mammalian Nav proteins. For example, one S6 sequenceaccording to the invention would be YMIFFMLVIFLGSFYLVNWILAVVAMAY (SEQ IDNO. 72).

As will be evident, functional recombinant sodium channel proteins maycontain multiple sequences of amino acids altered in the S6 segments ofdifferent domains. In preferred multiple-sequence embodiments, a firstamino acid sequence may be chosen from: WILAVVAMAY (SEQ ID NO. 36);LIL,WVVAMAY (SEQ ID NO.37); LICWVVAMAY (SEQ ID NO. 42); WICWVVAMAY (SEQID NO. 45), and LILAVWAMAY (SEQ ID NO. 59), and a second amino acidsequence chosen from: MYIAWILENF (SEQ ID NO. 60); MYIAIWLENF (SEQ ID NO.63); MYIACILENF (SEQ ID NO. 66); MYIAICLENF (SEQ ID NO. 67), andMYIAWWLENF (SEQ ID NO. 68). For obvious reasons, the two S6 sequenceswill not commonly be adjacent each other in primary sequence. In fact,in preferred embodiments, the sequences will be separated by at least400 amino acid residues, and, when the sequences reflect S6 segments indomains 1 and 4, they will be separated by at least 1000 residues.

In another aspect, the invention relates to a cell comprising arecombinant nucleic acid that encodes a mutant mammalian Nav1 protein.The mutant protein has a sequence as described above, and the cellexpresses sodium channels that exhibit a plateau current of greater than1 nanoamp. The process for transfecting a cell with an appropriatenucleic acid is well known in the art. The particular cell chosen fortransfection was the human embryonic kidney 1(EK293t) cell. Chinesehamster ovary cells are also well suited for this purpose. The person ofskill will be aware of cell lines that become known in the art for theirability to express proteins of the size (200 kDa) of the Nav protein,and these will be appropriate for transfection with the nucleic acids ofthe invention. Any eukaryotic cells which support stable replication ofplasmids containing nucleic acids encoding the mutant sodium channelsdescribed above may be used in practicing the invention. Non-limitingexamples of host cells for use in the present invention include HEK 293cells (American Type Culture Collection, Manassas, Va. (ATCC Cat. No.CRL-1573), CV-1/EBNA-1 cells (ATCC Cat. No. CRL 10478), HeLa cells,D98/raji cells, 293EBNA (Invitrogen, Cat. No. R62007), CV-I cells (ATCCCat. No. CCL 70) and 143B cells (ATCC Cat. No. CRL-8303). In addition,primary cultures of eukaryotic cells may be isolated from their tissueof origin and transfected with the appropriate expression vector.

One of the advantages of the substitutions of the present invention isthat, unlike all previously reported mutants of the Nav protein,substitution with W, F, Y or C in the carboxy terminal ten residues ofS6, when expressed at a useful level, results in a cell line that isviable (the leakage is not lethal to the cell), while at the same timethe cells exhibit a large enough sodium channel current to makemeasurement reliable.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA are used. Suchtechniques are well known and are explained in, for example, Sambrook etal., 2001, Molecular Cloning: A Laboratory Manual, Third Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: APractical Approach, Volumes I and II, 1985 (D. N. Glover ed.);Oligonucleotide Synthesis, 1984 (M. L. Gait ed.); Nucleic AcidHybridization, 1985, (Hames and Higgins, eds.); Transcription andTranslation, 1984 (Hames and Higgins, eds.); Animal Cell Culture, 1986(R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986, (IRL Press);Perbas, 1984, A Practical Guide to Molecular Cloning; the series,Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors forMammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold SpringHarbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wuand Grossman, and Wu, eds., respectively); Current Protocols inMolecular Biology, John Wiley & Sons, Inc. (1994), and all more currenteditions of these publications. Throughout this application, variousreferences are referred to within parentheses or square brackets. Thedisclosures of these publications in their entireties are herebyincorporated by reference as if written herein.

In the description that follows, certain conventions will be followed asregards the usage of terminology. The term “expression” refers to thetranscription and translation of a structural gene (coding sequence) sothat a sodium channel protein (i.e. expression product) havingbiological activity is synthesized. It is understood thatpost-translational modifications may remove portions of the polypeptidethat are not essential and that glycosylation and otherpost-translational modifications may also occur. The term“transfection,” as used herein, refers to the uptake, incorporation andexpression of exogenous DNA into a host cell by any means, and includes,without limitation, transfection of plasmids, episomes and othercircular DNA forms. The expression vector may be introduced into hostcells via any one of a number of techniques including but not limited toviral infection, transformation, transfection, lipofection or othercationic lipid based transfection, calcium phosphate co-precipitation,gene gun transfection, and electroporation. These techniques are wellknown to persons of skill in the art.

The term “sodium channel protein” refers to any protein that provides afunctional sodium channel in an excitable membrane. Known sodium channelproteins are the isoforms of the Nav family: Nav1.1 through Nav 1.9, Nav2.1 through Nav2.3 and Nav 3.1. [See Goldin Ann.N.Y.Acad.Sci 868:38-50(1999)]. For the present invention, type I Nav proteins (referredhereinafter as Nav 1 or Nav 1.x) are preferred, with Nav1.4 and Nav1.5being more preferred. The term “mutant sodium channel protein” or“recombinant sodium channel protein” refers to a recombinant proteinhaving the sequence of a Nav 1 protein, that is, Nav1.1 through Nav 1.9,in which from one to ten amino acids differ from the wild-type. Theperson of skill will of course recognize that in proteins of 2000 aminoacids, such as those of the Nav family, there can be innumerabledeletions, insertions and substitutions that do not affect the functionof the protein in any measurable way. Proteins having >90% homology to aprotein in the Nav family but containing deletions, insertions andsubstitutions that do not affect their function in providing a sodiumchannel are to be considered equivalents of the claimed mutants.Furthermore, because the genetic code is degenerate, more than one codonmay be used to encode a particular amino acid, and therefore, the aminoacid sequence can be encoded by any set of similar DNA oligonucleotides.With respect to nucleotides, therefore, the invention encompasses allthe DNA sequences containing alternative codons, which code for theeventual translation of the identical amino acid.

The term “plateau current” refers to the current measured in a singlecell 5 ms after activation by a sufficient voltage pulse to open thechannel. For cells expressing wild-type Nav1.4 and 1.5 the pulse is−60±10 mV and the plateau current is below 1 nA. For cells expressingmutant Nav1.4 and 1.5 according to the invention, the pulse can besomewhat higher (e.g. −70±10 mV) and the plateau current is above 1 nA.

The utility of the mutant Nav test system has been demonstrated withflecainide. Flecainide is one of several orally active class Icantiarrhythmic drugs. The primary target of flecainide is the cardiacNa⁺ channel, which is responsible for the upstroke of cardiac actionpotentials. Recently, flecainide has been found effective for patientswith the Long QT syndrome [Windle, et al., Ann. NoninvasiveElectrocardiol. 6(2):153-158 (2001)]. The state-dependent binding offlecainide with wild type and an exemplary inactivation-deficient sodiumchannel of the invention (rNav1.4-L435W/L437C/A438W) were compared inthe HEK293t expression system. Unlike the inactivation-deficient cardiacNav1.5 IFM/QQQ mutant of Grant et al. [Biophysical J. 79:3019-3035(2000)], the channel of the invention expressed well, which is evidentfrom the large sodium currents (>1 nanoamp following −50 mVstimulation), and we demonstrate below that flecainide binds rapidly andpreferentially with the open state but minimally with the resting state.This provides the basis for the first truly useful, high-throughputscreen for agents that may be used to treat various pathologicalconditions that manifest an increase in persistent late sodium currentsin the heart. Such agents include antiarrhythmic agents. To screen forsuch agents, one follows the procedure described in the experimentsdescribed below, and one simply replaces flecainide by a test agent.

The invention began from the hypothesis that an amino acid having abulky hydrophobic side chain, would disrupt or alter channel functionbecause of its large size. The disruption or alteration would occur if alarge hydrophobic amino acid were substituted for an amino acid thatcontacts or directly interacts with other parts of the channel protein.In addition, it was possible that the effects of several residues on thefast inactivation gating would be additive after multiple substitutions.The experiments below employed tryptophan (W) and cysteine (C) as theprototypic bulky, hydrophobic amino acids.

In practicing the method of the invention, a mammalian mutant Nav 1sodium channel protein is expressed in an appropriate cell. The cellexpressing the sodium channel of the invention is contacted with acompound to determine whether the compound has potential utility as ananti-arrhythmic agent.

Isolated nucleic acids comprising a nucleotide sequence that codes for amutant mammalian Nav 1 protein according to the invention may beobtained by methods known to one of skill in the art. Site-directedmutagenesis of DNA from appropriate cells, for example, heart and smoothmuscle, or cell line cultures of the appropriate species or tissue, isthen performed to obtain a nucleic acid encoding mutant sodium channelprotein as described above.

Isolation of DNA

DNA encoding a Na⁺ channel, in accordance with the instant invention,may be obtained by screening reverse transcripts of mRNA or cDNA fromappropriate cells or tissues, for example, CNS, skeletal muscle,denervated skeletal muscle, cardiac muscle, uterus, astrocytes or cellline cultures of the appropriate tissues, by screening genomiclibraries, or by combinations of these procedures. Screening of mRNA,cDNA or genomic DNA may be carried out with oligonucleotide probesgenerated from the nucleic acid sequence information of the sodiumchannel proteins disclosed herein.

An alternative means to isolate the gene encoding a Nav sodium channelprotein is to use polymerase chain reaction (PCR) methodology asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.

Site-Directed Mutagenesis

The QUIKCHANGE XL™ site-directed mutagenesis kit (Stratagene, La Jolla,Calif.) was used to create rat skeletal muscle Nav1.4 mutant clones aspreviously described (Wang and Wang, Biophys. J. 72:1633-1640, 1997;Wang and Malcolm, BioTechniques 26:680-682, 1999). Preliminarily, a wildtype rNav1.4-pcDNA1/Amp clone was generated to serve as the template formutagenesis. Briefly, a cDNA insert prepared from the wild type ratmuscle cDNA Nav 1.4, clone μl-2, (Genbank accession number M26643)(Trimmer et al., Neuron 3: 33-49, 1989) was cloned into the EcoRI siteof a pcDNA1/Amp vector (Invitrogen, Carlsbad, Calif.) to yield thevector rNav 1.4-pcDNA1/Amp. For mutagenesis, two complementary mutantoligonucleotides of 38-42 nucleotides in length (see Table 1) areannealed to the template DNA in separate tubes for 4 cycles of PCRreaction (94° C., 30 sec; 55° C., 1 min, 68° C., 23 min). TABLE 1Primers for Site-Directed Mutagenesis Clones rNav 1.4 437C438W/ rNav 1.4435W437C438W: 5′- ctcatcaatctgatctgctgggtggtgg (SEQ ID NO.: 73)ccatggcgtac - 3′ 5′- cctcatcaattggatctgctgggtggtg (SEQ ID NO.: 74)gccatggcgtac- 3′ Clones hNav 1.4 443C444W/ hNav 1.4 441W443C444W 5′-cctcatcaatctgatctgctgggtggtg (SEQ ID NO.: 75) gccatggcatatg - 3′ 5′-gctctttctacctcatcaattggatctg (SEQ ID NO.: 76) ctgggtggtggccatggcatatgc -3′ hNav 1.5 409C410W 5′- cctggtgaacctgatctgctgggtggtc (SEQ ID NO.: 77)gcaatggcc - 3′ 5′- ccttctacctggtgaactggatctgctg (SEQ ID NO.: 78) gg - 3′

The PCR reaction mix contains template DNA (0.4 ng/ul), primer (5ng/ul), KCl(10 mM), (NH₄)₂SO₄(10 mM), Tris-HCl (pH8.8) (20 mM),MgSO₄(2mM), TritonX-100 (0.1%), 0.1 mg/ml bovine serum albumin,deoxynucleotides mix (0.4 mM each), pfuTurbo DNA polymerase (0.05 U/ul).The stage 2 PCR reactions follow after mixing the two primer stage 1reactions into one and perform the following PCR reactions: 94° C., 30sec, 18 cycles of (94° C., 30 sec, 55° C., 1 min; 68° C., 23 min). Thein vitro synthesized DNA is digested with DpnI at 0.2 U/ul at 37° C. forone hour. One μl of the DpnI treated DNA is transformed into XL-Goldultracompetent cells (Strategene, La Jolla Calif.), plated on Ampicillin(50 ug/ml) LB plates. Bacterial colonies are picked into LB containingAmpicillin at 50 ug/mlDNA. The mutation frequency is 25-100%, that is,you will obtain at least one mutant if you sequence 4 clones.

To minimize the possibility that unique phenotypes are due to unwantedmutations, independent clones of rNav1.4-L435W/L437C/A438W andrNav1.4-L437C/A438W as well as additional homologous L435W/L437C/A438Wclones from human isoforms (hNav1.4 and hNav1.5) were created.Preliminary results showed that all of these independent and homologousclones displayed comparable phenotypes to those of rNav1.4 counterparts.DNA sequencing near the mutated site was performed to confirm themutations.

Transient Transfection

Transfection methods are well known in the art. In one embodiment, humanembryonic kidney (HEK293t) cells were grown to ˜50% confluence in DMEM(Gibco) containing 10% fetal bovine serum (HyClone, Logan Utah), 1%penicillin and streptomycin solution (Sigma, St. Louis, Mo.), 3 mMtaurine, and 25 mM HEPES (Gibco). HEK293t cells were then transfectedwith cloned Na⁺ channels, either wild type or mutant, by a calciumphosphate precipitation method in a TI-25 flask (Cannon andStrittmatter, 1993). A reporter plasmid CD8-pih3m and cDNA clone in thepcDNA1/amp vector (Invitrogen, San Diego, Calif.) were prepared in 250mM CaCl₂, added to a test tube containing 0.36 ml of Hanks' balancedsalt solution and incubated at 22° C. for 20 min. The DNA solution wasthen dripped over a cell culture (30-50% confluence) containing 7 ml ofDMEM. The transfected cells were trypsinized and replated 15 h later toan appropriate density in 35-mm tissue culture dishes containing 2 ml offresh DMEM. Transfected cells were maintained at 37° C. in a humidified5% CO₂ incubator, and used after 1-4 days. Transfection-positive cells,which were identified by binding to immunobeads (CD8-Dynabeads, Dynal,Lake Success N.Y.) coated with a monoclonal antibody specific for CD8antigen, were selected for patch-clamp experiments.

Transfection of wild type rNav1.4-pcDNA1/Amp or mutant clones (5-10 μg)along with β1-pcDNA1/Amp (10-20 μg) and reporter CD8-pih3m (1 μg)generated sufficient sodium channel expression for later currentrecording.

Measurement of Na⁺ Current

Whole-cell configuration was used to record Na⁺ currents according tothe method of Hamill et al. [Pflugers Arch. 391:85-100 (1981)].Borosilicate micropipettes (Drummond Scientific Company, Broomall, Pa.)were pulled with a puller (P-87, Sutter Instrument Company, Novato,Calif.) and heat polished. Pipette electrodes contained 100 mM NaF, 30mM NaCl, 10 mM EGTA, and 10 mM HEPES adjusted to pH 7.2 with CsOH. Thepipette electrodes had a tip resistance of 0.5 to 1.0 MΩ. Accessresistance was 1-2 MΩ and was further reduced by series resistancecompensation. All experiments were performed at room temperature (22-24°C.) under a Na⁺-containing bath solution with 65 mM NaCl, 85 mM cholineCl, 2 mM CaCl₂, and 10 mM HEPES adjusted to pH 7.4 withtetramethylammonium hydroxide. Residual outward currents were evident insome cells at voltages ≧+30 mV; these currents were present inuntransfected cells and were insensitive to tetrodotoxin. These residualcurrents were not subtracted from the measurements. Whole-cell currentswere measured by an AXOPATCH 200B™ (Axon Instruments, Foster City,Calif.) or an EPC-7 (List Electronics, Darmstadt/Eberstadt, Germany),filtered at 3 kHz, collected, and analyzed with pClamp8 software (AxonInstruments). Leak and capacitance were subtracted by the patch clampdevice and further by the leak subtraction protocol (P/−4). Cells wereheld at −140 mV for functional characterizations. Voltage error was <4mV after series resistance compensation. An unpaired Student's t testwas used to evaluate estimated parameters (mean±SEM or fitted value±SEof the fit); P values of <0.05 were considered statisticallysignificant.

Gating properties of W substitutions within the C-terminus of D1S6 inNav1.4 Na⁺ channels were examined. To characterize the effects of Wsubstitutions, we measured Na⁺ currents of D1S6 W-substituted mutantchannels at various voltages. Each of residues 19 to 28 (the carboxyterminus of S6) was replaced by W.

FIGS. 2A and B show the superimposed current families of Nav1.4 wildtype (A) and mutant Nav1.4-A438W (position 22 at D1S6) (B),respectively, cotransfected with β1 subunit. Activation threshold wasaround −50 mV for wild type and around −60 mV for A438W mutant channels.Families of Na⁺ currents for wild type (A) and Nav 1.4-A438W mutant (B)were evoked by 5 ms pulses from the holding potential (−140 mV) tovoltages ranging from −120 to +50 mV in 10-mV increments. The currenttraces evoked by a pulse to −50mV and to +50 mV are labeled. Normalizedmembrane conductance (g_(m))(C) was determined from the equationg_(m)=I_(Na)/E_(m)−E_(Na)), where I_(Na) is the peak current, E_(m) isthe amplitude of the pulse voltage, and E_(Na) is the reversalpotential, and plotted against the pulse voltage. Plots were fitted witha Boltzmann function, which yielded the midpoint voltage (V_(0.5)) andslope (k) for wild-type (open circles, n=5) of −32.0±0.9 nV and 8.7±0.8nV, respectively, and −38.8±1.2 mV and 13.1±1.1 mV for rNav 1.5-A438W(closed circles, n=6)(FIG. 2C). The W mutant showed an apparent leftwardshift of −6.8±2.1 mV (n=6).

FIG. 3 is a summary of effects of W- and selected C-mutations atC-termini of D1S6 and D4S6 on activation and inactivation gating. In theleft panel is a vertical representation of amino acid sequences of D1S6(top), D4S6 (middle), and double and triple mutants (bottom). Allmutants and wild-type Na⁺ channels were co-transfected with the β1subunit. Activation Shift: the bar graph shows the differences involtage for the half-maximal activation (V_(0.5)) of the wild-type andmutant Na⁺ channels. The V_(0.5) values (mean±SEM) were obtained fromthe Boltzmann fits of normalized conductance versus voltage plots asdescribed above for FIG. 2. Significantly, the estimated reversalpotential (E_(Na)) remained about the same in these mutants. Bars ingray indicate single C-substitution. Non-expressing mutants are notedwith an X. Slope Effect (Activation, Inactivation): this bar graph showsthe differences in k values for the wild-type and mutant channels. The kvalues (mean±SEM) were obtained from Boltzmann fits of I/V plots(activation) and from Boltzmann fits of steady-state inactivation plots(inactivation), as described for FIG. 2 and FIG. 5, respectively.Inactivation Shift: the bar graph shows the differences in voltage forthe half-maximal inactivation (h_(0.5)) of the wild-type and mutant Na⁺channels. The h_(0.5) values (mean±SEM) were obtained as described forFIG. 5. All values were derived from n=4-6, except E1592W with n=3. Anasterisk (*) indicates that the value is statistically different fromthat of the wild type (p<0.05). Except for L435W and I436W, all otherD1S6 mutants W displayed a leftward shift. L437W shifted leftward by asmuch as −22.1±2.0 mV (n=5). The slope factor for each W mutant showedeither no significant change or became less steep.

Another noticeable change in gating after A43 8W substitution was thenon-inactivating currents maintained at the end of the pulse (FIG. 2Bvs. FIG. 2A). To quantify this difference between wild type and mutantchannels we measured the amount of the non-inactivating maintainedcurrent near the end of 5-ms+50 mV pulse. The wild-type current decayedrapidly and reached its steady-state level within 2-3 ms and 2.9±0.8%(n=5) of currents were maintained under these experimental conditions.In contrast, Nav1.4-A438W mutant showed conspicuous maintained currentsunder identical conditions. As measured near the 5-ms time, 31.9±4.2%(n=6) of currents were non-inactivating. Thus, a substitution of W atposition 22 of D1S6 impairs the Na⁺ channel fast inactivationsignificantly (P<0.001).

In FIG. 4, the relative maintained currents in various W- andC-mutations of C-termini of D1S6 and D4S6 are shown, with the relativenon-inactivating components of all mutants at D1S6 on the left. Cellswere cotransfected with β1 subunit; non-expressing mutants are indicatedby an X. The fraction of non-inactivating current was determined as theaveraged current amplitude near 5 ms after the +50 mV pulse (e.g., FIG.2) divided by the peak current. Error bars indicate standard error.Asterisks indicate significant differences from the wild-type channelsas determined by a t test (p<0.05, n=4-6). Dotted line indicates thevalue of wild-type. Bars in gray indicate single C-substitution.

The two other W mutants with impaired fast inactivation are L435W (FIG.4; the fraction of non-inactivating current=0.10±0.02, n=5, P<0.05;position 18) and L437W (0.055±0.017, n=5, P=0.20; position 21).

Using various conditioning pulses from −160 mV to −15 mV, we furthercharacterized the steady-state inactivation of the mutant channels. FIG.5A and B show Na⁺ currents of the wifld type and Nav1.4-A439W mutant,respectively, cotransfected with β1 subunit, evoked by a 5-ms test pulseto +30 mV. Test pulses were preceded by 100 ms conditioning pulsesranging from −160 mV and −15 mV in 5-mV increments. Smallnon-inactivating inward currents appeared at conditioning voltages ≧−60mV.

Peak currents were measured, normalized with respect to the peak currentat −160 mV and plotted against conditioning voltages. In FIG. 5C,normalized Na⁺ current availability (h∞) of wild type (open circle, n=5)and rNav1.4-A438W (closed circle, n−5) were plotted as a function of the100 ms conditioning pulse voltage. The data were fitted with a standardBolzmann equation. The fitted midpoint(h_(0.5)) and slope factor (k) forwild-type were −73.4±0.1 mV and 5.0±0.1 mV, respectively, and −89.0±0.3mV and 6.0±0.2 mV for Nav1.4-A438W, respectively. The shift in theΔV_(0.5) is shown in FIG. 3 (top on right side) along with the shift inthe slope factor, the Δk value.

The residues from position 19 to 26 in D4S6 were also substituted withtryptophan. FIGS. 6A and B show the current voltage relationship andsteady state inactivation measurement of mutant Nav1.4-I1589W,respectively. In 6A superimposed Na⁺ current traces were evoked by 5-mspulses from the holding potential (−140 mV) to voltages ranging from−120 to +50 mV in 10-mV increments. For 6B, superimposed currents wereevoked by a 5-ms test pulse to +30 mV preceded by 100-ms conditioningpulses ranging from −160 mV to −15 mV in 5-mV increments.

Normalized Na⁺ conductance (g_(m)), as shown in FIG. 6C, was derivedfrom A as described above for FIG. 2, plotted against voltage, andfitted with a Boltzmann function. The fitted midpoint voltage (V_(0.5))and slope (k) of the function for wild-type (open circles, n=5) were−32.0±0.9 and 8.7±0.8, respectively, and −19.0±1.0 and 12.6±0.9,respectively for rNav1.4-I1589W (closed circles, n=6). Normalized Na⁺current availability (h_(∞)) of wild-type (open down triangle, n=5) andNav1.4-I1589W (closed down triangle, n=5) were derived from (B) asdescribed for FIG. 5, and plotted as a function of conditioning voltage.Plots were fitted with the Boltzmann function. The midpoint (h_(0.5))and slope factor (k) for wild-type were −73.4±0.1 and 5.0±0.1,respectively, and −66.5±0.2 and 5.7±0.2, respectively, for theNav1.4-I1589W. Cells were cotransfected with β1 subunit.

Again there were significant non-inactivating currents maintained at theend of test pulse for I1589W. The relative amounts of the maintainedcurrents of all mutants at D4S6 are listed in FIG. 4 (middle section)along with D1S6 mutants. The activation of I1589W was shifted rightwardby 13.0±1.9 mV (n=6) and the steady state inactivation was shiftedrightward by 6.8±0.3 mV (n=5). These changes in gating parameters of allD4S6 mutants are listed in FIG. 3 (middle section). Two W mutantchannels (I1589W and I1590W) appeared to have significantly impairedfast inactivation. Two mutants, I1587W and A1588W, expressed Na⁺currents below 1 nA in this expression system.

There is not a clear relationship between the size of the residue in thenative amino acid sequence and the degree of impairment in fastinactivation. FIGS. 7A and B show the current families of A438C andI1589C, respectively. Superimposed Na⁺ current families of Nav1.4-A438C(7A) and Nav1.4-I1589C (7B) were evoked by 5-ms pulses from holdingpotential of −140 mV to voltages ranging from −120 to +50 mV in 10-mVincrements. In FIG. 7C, normalized membrane conductance (g_(m)) isplotted versus the amplitude of the 5-ms voltage step. G_(m) wasdetermined as described for FIG. 2 above, plotted against the membranevoltage, and fitted with a Boltzmann function. The fitted midpointvoltage (V_(0.5)) and slope (k) of the function for wild-type (opencircles, n=5) were −32.0±0.9 and 8.7±0.8, respectively, −25.9±0.7 and8.3±0.6 for Nav1.4-A438C (closed square, n=5), and −39.9±1.1 and10.9±0.9 for Nav1.4-I1589C (closed triangle, n=6). Cells werecotransfected with β1 subunit.

A438W and I1590W exhibited significantly impaired fast inactivation butA438C and I1590C do not (FIGS. 2, 4). In contrast, I1589C displayedimpaired fast inactivation similar to that of I1589W (FIG. 4). This lackof direct correlation in volume suggests that either allosteric effectsoccur after amino acid substitutions (i.e. the sulfhydryl undergoesreaction with a nearby bulky, hydrophobic nucleophile) or these residuesmay specifically and directly interact with other parts of channelstructure, such as the inactivation gate.

Gating properties of double and triple substitutions of residues withinD1S6 and D4S6 were also tested. Selected residues (L435, L437, A438,I1589, I1590) were multiply substituted. All mutants were co-transfectedwith the β1 subunit.

Superimposed Na⁺ current families were evoked by 5-ms pulses fromholding potential of −140 mV to voltages ranging from −120 to +50 mV in10-mV increments for mutants A438W/I1589W (A), L437C/A438W (B),L435W/L437C/A438W (C) AND I1589W/I1590W (D). FIGS. 8A, B, C and D showthe current families of A438W/1589W, L437C/A438W, L435W/L437C/A438W, andI1589W/I1590W, respectively.

Several multiple-substituted mutants expressed a high level of Na⁺currents comparable to that of wild type. There were two distinct typesof phenotypes from these mutants. One type showed supra-additive effectson the fast inactivation and the other showed sub-additive effects. Theresults thus demonstrate that it is feasible to create fast-inactivationdeficient mutants that express well in a mammalian expression system.

When the fast inactivation was hampered by pronase or by site-directedmutagenesis, slow inactivation gating not only remained functional butalso was accelerated considerably. This inverse relationship suggeststhat the fast inactivation and slow inactivation gating have distinctidentities and yet these two gating processes are somehow coupled. Todetermine whether such inverse relationship holds true in S6 mutantswith severely impaired fast inactivation, we therefore measured the slowinactivation gating with a 10-s conditioning pre-pulse at variousvoltages.

To induce slow inactivation, we applied conditioning prepulses rangingfrom −180 mV to 0 mV with a duration of 10 s. After a 100-ms interval at−140 mV, Na⁺ currents were evoked by the delivery of a +30 mV testpulse. (A) Peak Na⁺ currents were normalized to the correspondingcurrent obtained with a prepulse to −180 mV and plotted againstconditioning prepulse potential. Data were fitted with a Boltzmannfunction. The fitted V_(0.5) values and k (slope factor) values from theBoltzmann functions were (in mV) −51.1±0.5 and 9.7±0.4, respectively,for wild-type (open circle, n=5); −49.9±0.1 and 5.0±0.1, respectively,for L435W/L437C/A438W (closed circle, n=5); −45.2±0.3 and 5.8±0.2,respectively, for L437C/A438W (closed up triangle, n=5); −45.2±0.3 and6. 8±0.3, respectively, for A438W/I1589W (closed down triangle, n=6);and −50.0±0.9 and 9.5±0.8, respectively, for I1589W/I1590W (closeddiamond, n=5). The final steady-state non-inactivated values (in %) were57.5±0.4 (wild-type), 3.3±0.4 (L435W/L437C/A438W), 12.3±0.6(L437C/A438W), 11.6±0.8 (A438W/I1589W), and 3.4±1.7 (I1589W/I1590W). Allmutants are co-transfected with the β1 subunit. (B) Development of slowinactivation. For the development of slow inactivation, the prepulseduration at +30 mV was varied ranging from 0 to 10 s. The peak currentat the test pulse of +30 was measured and normalized to the initial peakamplitude without a prepulse, and then plotted against the prepulseduration. The data were fitted by a single-exponential function. The rvalues (and final steady-state Y₀ values) for wild-type,L435W/L437C/A438W, L437C/A438W, A438W/I1589W, and I1589W/I1590W are4.8±0.3 s (51.6%), 0.72±0.01 s (3.0%), 0.50±0.01 s (16.6%), 0.64±0.02 s(10.5%), and 0.81±0.02 s (16.6%), respectively.

With a gap of 100 ms at −140 mV, which allowed channels to recover fromtheir fast inactivation but not from their slow inactivation, weobserved that 57.1±3.6% (n=5) of wild type Na⁺ currents were slowinactivated at 0 mV for 10 s (FIG. 9A; open circles). In contrast,almost all L435W/L437C/A438W mutant channels were slow-inactivated (FIG.9A, closed circles) at 0 mV under these experimental conditions. Itappeared that this enhanced slow inactivation is in part due to theenhanced forward rate constant as shown in FIG. 9B. Multiple-substitutedmutants with enhanced slow inactivation were inactivated with a ratherrapid rate, with a time constant of <1 sec at +30 mV (vs. 4.8 s for wildtype). It is noteworthy that slow inactivation in wild type channelsdoes not reach its steady state with a 10-s conditioning pulse even at+30 mV (FIG. 9B). Nonetheless, this pulse protocol allowed us todetermine which mutants exhibit altered slow inactivation significantly.In general, we observed that mutants with the most impaired fastinactivation (L435, L437, A438, I1589, I1590) were those with enhancedslow inactivation. In particular, the multiple-substituted mutants, suchas L437C/A438W and L435W/L437C/A438W, with the most impaired fastinactivation also had the most enhanced slow inactivation.

The study demonstrates that most mutants with a single W substitution atthe C-terminus of D1S6 and D4S6 express observable Na⁺ currents inHEK293t cells. Substitutions with W in this region alter Na⁺ channelactivation, fast inactivation, and/or slow inactivation gating invarying degrees, dependent on the position of the substitution. Fivepositions (L435, L437, A438, I1589, and I1590) appear to be closelyassociated with fast inactivation gating. Two mutants (L437C/A438W andL435W/L437C/A438W) with multiple W or C substitutions exhibit minimalfast inactivation. Interestingly, all mutants with impaired fastinactivation display an enhanced slow inactivation phenotype. The orderof the level of the maintained current in D1S6/D4S6 residues is asfollows: A438W (31.9%)>I1590W (20.0%)>I1589C (18.6%)>I1589W(14.5%)>L435W (9.5%)>L437C (6.7%)>L437W (5.5%)>wild type (2.9%). Theamount of maintained current of L437W and L437C is higher than that ofthe wild type but it did not reach the level of statisticalsignificance. In comparison, Nav1.2-L421C (equivalent to Nav1.4-L437C)has a maintained current of ˜10% of the peak current, significantlyhigher than its wild type (˜2%). Evidently, differences in the amount ofmaintained currents exist between mutants derived from differentisoforms. The magnitude of the slow inactivation and the fastinactivation appear to have an apparent inverse relationship. Thefraction of slow-inactivated channels followed the order I1590W(85.9%)>I1589W (80.6%)>A438W (76.6%)>L435W (75.5%)>L437W (58.7%)>wildtype (43.0%) at 0 mV (FIG. 10); these mutants happened to be fiveresidues with impaired fast inactivation. Furthermore,L435W/L437C/A438W, L437C/A438W, and A438W/I1589W mutants with multiplesubstitutions have minimal fast inactivation, and they showsignificantly enhanced slow inactivation (96.8%, 93.2%, and 88.3%,respectively).

FIG. 10 shows a coarse correlation between fast and slow inactivationgating. The relative level of slow inactivation for D1S6 mutants (left),D4S6 (middle), and double and triple mutants (right) is shown in FIG.10A. Non-expressing mutants are noted with an X. Values representmean±S.E. of peak current elicited by a 5 ms test pulse to +30 mVpreceded by a 10-s conditioning pulse to 0 mV and a 100 ms interval at−140 mV as described in FIG. 9A. Asterisks indicate significantdifferences from the wild-type channels as determined by a t test(p<0.05). Dotted line indicates the value of wild-type. Bars in grayindicate single C-substitution. The fraction of slow-inactivated currentvs. the fraction of non-inactivating current of the individual mutant isshown in FIG. 10B. Data for wild type and mutants were taken from FIG. 4for x axis and from FIG. 10A for y axis. The mutants are labeled. Thesolid line is the linear fit of the complete data set with a correlationcoefficient (r) of 0.61. Mutants with impaired fast inactivation(fraction of non-inactivating current >5%) are shown at the right-handside of the dashed line; all of them show enhanced slow inactivation.

It is surprising to find that the mutants with minimal fast inactivationexpress as well as the wild type in mammalian cells. Previous reports inthe literature indicated that, unlike wild-type Na⁺ channels, variousfast-inactivation deficient mutants at the IFM motif expressed poorly inHEK293 expression system under the same conditions. Theinactivation-deficient S6 mutants of the invention are useful tools forfuture studies, including the establishment of permanent cell lines, thescreening for potent open-channel blockers that block persistent opening(e.g. anti-arrhythmic agents), the ion permeation in the persistent openchannel, and the detailed studies on direct interactions between drugsand the open channel.

Studies with rNav1.4-L435W/L437C/A438W demonstrated that flecainidebinds rapidly and preferentially with the open state but minimally withthe resting state. Flecainide is very effective in blocking persistentlate Na+ currents as evident from its strong time-dependent block ofmaintained currents during prolonged depolarization. Flecainide bindingwith the inactivated state is considerably slower than that with theopen state by orders of magnitude. Once the channel is blocked byflecainide, the inactivation gate may stabilize receptor-flecainidecomplex, as the dissociation rate of flecainide is extremely slow andrequires ≧1,000 s (˜17 minutes) for the full recovery.

We first measured the Na+ current family at voltages ranging from −60 mVto +50 mV. We then applied 30 mM flecainide externally and measured theNa+ current at +50 mV for 5 ms at a 30-s interval. About 50% of the peakcurrents were inhibited after flecainide block reached its steady state,usually within 5-7 minutes. We then re-measured the Na+ current familyin the presence of 30 mM flecainide and found that the current kineticsremained unchanged and the conductance/voltage curves remainedcomparable with or without flecainide.

The steady-state inactivation of Na+ channels was measured by a standardtwo-pulse protocol at a test pulse of +30 mV with various conditioningpulses ranging from −160 mV to −15 mV for 100 ms, with and withoutflecainide. There was a leftward shift of a few mV and the slope factorappeared less steep.

We applied a voltage scanning protocol ranging from −180 mV to −10 mV todetermine whether distinct binding affinities of flecainide exist inrNav1.4 Na+ channels. This pulse protocol consisted of a conditioningpulse at various voltages with a 10-ms duration intended for drugbinding. It was originally designed to test if inactivated channels havehigher “saturable” affinities than resting channels for localanesthetics.

Conditioning prepulses ranging from −180 mV to −10 mV were applied for10 s. After a 100-ms interval at −140 mV, Na⁺ currents were evoked by atest pulse at +30 mV for 5-ms. Currents recorded before (open circle,n=6) and after 30 μM flecainide (closed circle, n=6) were normalized tothe current obtained at the −180 mV conditioning pulse and plottedagainst the conditioning voltages. Flecainide data were thenrenormalized at each voltage with respect to the control value withoutflecainide. Data were fitted with a Boltzmann function(1/[1+exp((V_(0.5)-V)/k_(E))]). The average V_(0.5) value and k_(E)(slope factor) value for the fitted functions were (in mV) −47.9±1.1 and8.6±0.9, respectively, for control and −57.3±2.7 and 17.6±2.1,respectively, for flecainide.

FIG. 11 shows that flecainide at 30 μM blocks resting channels at aconstant level from −180 mV to −100 mV. The block increases continuouslyfrom −80 mV to −20 mV.

We measured the dose-response curve of flecainide with a 10-sconditioning pulse at −140 mV, −70 mV, and −20 mV, again at a 30-sinterval for the flecainide to reach steady state. Binding at thesethree voltages for local anesthetics generally represents the resting,closed/inactivated, and open/inactivated affinities, respectively. Themeasurements at −20 mV, −70 mV, and −140 mV provided IC₅₀ values of13.4±0.3, 21.2±0.4, and 31.9±3.0 mM, respectively. The differencebetween the high and low flecainide affinities is less than 3-fold. Inthe case of cocaine, the difference between the resting and inactivatedblock is 28-fold (250 mM vs. 9 mM).

One possibility for the rightward shift of the voltage dependence offlecainide block is that the inactivated channels interact with the drugrather slowly. This appeared to be the case for the development of theinactivated block at −50 mV with a time constant of 10.9±1.3 s. Oncedeveloped, the recovery from this inactivated block by flecainide at 100mM was also very slow with a time constant of >100 s, as if flecainidewas trapped within the channel. Unexpectedly, the amplitude of the Na+currents continued to increase during this recovery period and reached alevel that is 78% to the control amplitude without flecainide. A sameslow time course also occurred after the block was developed at +30 mV.Thus, both closed/inactivated and open/inactivated block by flecainiderecovered nearly to the level about ˜80% of the control value with thesame slow time course. These results indicated that the resting block at−140 mV by flecainide is much less than the block normally measured atthe 30-s interval. The estimated IC₅₀ for the resting block offlecainide in wild-type channels is 355 mM at −140 mV.

To test whether flecainide interacts with the open state of Na+ channelswe therefore applied repetitive pulses for channel activation.Repetitive pulses at 5 Hz for 60 pulses elicited additionaluse-dependent block of flecainide by ˜50%. The total duration ofdepolarization was 1.44 s (0.024×60). Therefore, it appears thatflecainide binds to the open state of Na+ channels with a faster ratethan that of the inactivated state since the similar long pulse did notelicit a comparable use-dependent block. This being true, keeping thechannel open persistently during depolarization enhances the block offlecainide.

To determine interactions between flecainide and the open statedirectly, we used inactivation-deficient rNav1.4-L435W/L437C/A438Wmutant channels of the invention. This mutant channel inactivatedminimally during depolarization; instead, a substantial fraction of peakcurrent was maintained. FIG. 12 shows the current families before andafter flecainide at various concentrations ranging from 0.1 to 30 μM. At+50 mV, there was a strong time-dependent block of the maintained Na+currents. This result therefore provides the direct evidence thatflecainide binds preferentially with the open state of the Na+ channeland indicates that Nav mutants of the invention can be used as tools fordetailed studies on sodium channel block.

We generated non-inactivating Na+ currents using a test pulse of +30 mVand then measured the time-dependent block of flecainide at variousconcentrations. FIG. 13 shows the decay phase of the Na⁺ current. Thedecay phase of the Na+ current could be well fitted with a singleexponential function and the time constant (τ) was inverted and plottedagainst the corresponding concentration. Data were fitted with a linearregression y=14.9×+12.16 (solid line). The on-rate and off-rate constantof flecainide with the open channel are estimated to be 14.9 μM⁻¹s⁻¹(the slope factor) and 12.2 s⁻¹ (y-intercept), respectively. Thecalculated dissociation constant yields 0.81 μM.

The IC₅₀ values for the open (estimated block at the end of the pulse)and the resting block (estimated block at the peak current) were0.61±0.07 μM and 208.3±16.9 μM, respectively. In contrast, with aconditioning pulse at −50 mV for 10 s, the IC₅₀ was 4.1±0.1 μM(estimated block at the peak current) or about 7-fold less potent thanthat of the open channel block. This suggests channel opening isrequired for the high-affinity block of flecainide. With limited channelopening around activation threshold of −50 mV, the flecainide affinityis not as high as that of the open channel block.

Repetitive pulses at 5 Hz demonstrate that flecainide produces anadditional use-dependent block in the peak current amplitude. Itappeared that this rapid phase of the use-dependent block was causeddirectly by the time-dependent block of the non-inactivating currentduring the pulse. This time-dependent block recovers little during the200-ms interpulse at −140 mV. There was also a slow inhibition of peakcurrents during repetitive pulses in inactivation-deficient channelseven without flecainide.

From the foregoing results one may conclude that: (1) Flecainide blockof the wild-type Na+ channel developed after channel activation has avery slow recovery time course, up to 10,000 s (or ˜17 minutes) at theholding potential of −140 mV. Any pulse protocol that activates Na+channels at a frequency as low as one per 30 s will significantlyperturb the degree of flecainide block. (2) The resting and open channelaffinities differ by ˜500-fold in the inactivation-deficient mutantchannels of the invention (0.61 μM vs. 307 μM, respectively). (3) Therecovery from the open channel block by flecainide is relatively fast at−140 mV, with a time constant of 11.2 s in inactivation-deficient mutantchannels, or several orders faster than that with intact fastinactivation in wild-type Na+ channels.

Flecainide appears to interact with the resting state of Na+ channelsrather weakly. At 100 mM flecainide blocks only about ˜20% of peak Na+currents if the cell is not stimulated repetitively in 1,000 s. Thecalculated IC₅₀ for flecainide block is therefore about 400 μM. It willbe difficult to measure this value directly in a single cell having awild-type Nav protein at various concentrations, since only one testpulse per 17 minutes can be applied for such dose-response assay. Incontrast, the IC₅₀ for flecainide block of inactivation-deficient mutantNa+ channels of the invention can be estimated directly from the peakcurrent amplitude, which yields 307±19 μM for the resting block.

Flecainide appears to be a rather pure open channel blocker with minimalinteractions with resting state. Flecainide has been shown to bebeneficial for the treatment of a number of genetic diseases withmutations on the Na+ channel (e.g., Brugada et al., 1999; Windle et al.,2001). Many of these defective channels exhibit persistent late Na+currents lasting hundreds of milliseconds during prolongeddepolarization, such as in the cases of DKPQ (Bennett et al., 1995) orhyperkalemic periodic paralysis (Cannon et al., 1991). Recently,Nagatomo et al (2000) found that flecainide preferentially blocks thelate Na+ currents in the DKPQ mutant. The results above demonstrate thatflecainide indeed blocks the maintained persistent Na+ currentseffectively and rapidly. The therapeutic plasma concentration offlecainide is 0.4 to 2 μM as an antiarrhythmic agent. At thisconcentration range, a substantial fraction of the persistent latecurrent should be blocked by flecainide, which exhibits an IC₅₀ of 0.61μM for the open channel. The persistent late currents are likely morevulnerable to flecainide block as the peak currents are rapidlyinactivated and may not be blocked in time. The open-channel selectiveblockers, such as flecainide and pilsicainide have broader applicationsfor various pathological conditions that manifest an increase inpersistent late Na+ currents in the heart (Saint et al., 1992), in brain(Crill, 1996) or in muscle (Cannon, 1996) and the search for improvedagents to treat these pathological conditions is greatly advanced by thescreen of the present invention.

Stable Expression of hNav1.4-L443C/A444W Inactivation-Deficient MutantNa⁺ Channels in HEK293 Cells

The advantages of a permanent mammalian cell line expressinginactivation-deficient mutant Na⁺ channels are two-fold. First, apermanent cell line would simplify in vitro studies ofhNav1.4-L443C/A444W mutant Na⁺ channels including biophysical studies ofthe persistent open channel and pharmacological characterizations ofdrug effects on late Na⁺ currents. Second, the cell line may be utilizedto screen potent open-channel blockers using an automated parallelpatch-clamp system (e.g., PatchXpress or Ionworks HT) [Sanguinetti andBennett, 2003].

Previous attempts to establish IFM/QQQ inactivation-deficient Na⁺channels in this cell line suggested that sodium overload fromspontaneous channel openings inhibited channel expression [Grant et al.,2000]. In contrast, a relatively high level of expression ofinactivation-deficient Na⁺ channels can be attained over several monthsin the stably transfected HEK293 cells of the present invention. Severalfactors may favor the expression of hNav1.4-L443C/A444W mutant Na⁺channels in our HEK293 cell line: the enhanced slow inactivation,rightward shifts in activation gating, and/or posttranslationalmodifications.

After transient transfection of an hNav1.4-L443C/A444W clone, HEK293cells exhibited robust inactivation-deficient Na⁺ currents. We thereforeattempted to establish a stable HEK293 cell line expressing a high levelof persistent late Na⁺ currents.

For preparation of the transfected cells of the present invention,full-length hNav1.4 cDNA inserted in the pRc/CMV vector was obtainedfrom Dr. Theodore Cummins (Indiana University, Indianapolis, Ind.).Mutagenesis of hNav1.4-L443C/A444W cDNA was achieved as described abovefor the creation of rat skeletal muscle Nav1.4 mutant clones. Themutations occurred at position 21-22 of the D1S6 C-terminus [Wang etal., 2003a].

Human embryonic kidney (HEK293) cells were maintained at 37° C. in a 5%CO₂ incubator in DMEM (Life Technologies, Inc., Rockville, Md.)containing 10% fetal bovine serum (HyClone, Logan, Utah), and 1%penicillin and streptomycin solution (Sigma, St. Louis, Mo.). HEK293cells were transfected with the mutant clone in pRc/CMV vector alongwith a rat β1 subunit in pcDNA1 by a calcium phosphate precipitationmethod [Wang et al., 2004]. Transfected HEK293 cells were treated with 1mg/ml G-418 (Invitrogen, Inc.) during selection in 100-mm culturedishes. The selection DMEM medium contained no penicillin orstreptomycin. Individual G-418 resistant colonies were isolated usingglass cylinders (i.d.=6 mm) 2 weeks after transfection. Isolatedcolonies were trypsinized and replated in 35-mm culture dishes each witha gelatin-treated coverslip. The coverslip was removed from the dishafter 2-3 days and ≧5 cells from each coverslip were assayed under thewhole-cell configuration.

Five positive colonies with inactivation-deficient currents wereexpanded and frozen individually. One colony was later reestablished andmaintained in Ti-25 flasks for studies described here. As expected, thiscolony did express mRNA of the Nav1.4 α-subunit, but not β1-subunit asdetermined by PCR. Evidently, without neomycin/G418-resistance gene theβ1-pcDNA1 vector was excluded during G-418 selection.

One colony expressing Na⁺ currents was selected from a total of 5positive colonies for further studies. Stably transfected cells werereestablished from a frozen vial and were maintained with continuouspassages weekly over a period of four months in Ti-25 flasks. It was notnecessary to include G-418 in the media once the cell line wasreestablished.

The whole-cell configuration of a patch-clamp technique [Hamill et al.,1981] was used to study Na⁺ currents in stably transfected HEK293 cellsat room temperature (22±2° C.). Electrode resistance ranged from 0.5 to1.0 MΩ. Command voltages were elicited with pCLAMP8 software anddelivered by Axopatch 200B (Axon Instrument, Foster City, Calif.). Cellswere held at −140 mV and dialyzed for ˜10-15 min before currentrecording. Most of the capacitance and leak currents were cancelled witha patch-clamp device and by P/−4 subtraction. Liquid junction potentialwas not corrected. Peak currents at +50 mV were ˜2-20 nA for themajority of cells. Access resistance was about 1-2 MΩ; series resistancecompensation of >85% typically resulted in voltage errors of ≦4 mV at+50 mV. All current measurements were performed at +30 mV or +50 mV forthe outward Na⁺ currents. Such recordings allowed us to avoid thecomplication of series resistance artifacts and to minimize inward Na⁺ion loading (Cota and Armstrong, 1989). Curve fitting was performed byMicrocal Origin (Northampton, Mass.).

Flecainide-HCl, amiodarone-HCl, and mexiletine-HCl were purchased fromSigma (St. Louis, Mo.). (S)-Propafenone-HCl and batrachotoxin (BTX) weregenerous gifts from Dr. Wolfgang Lindner (Graz, Austria) and Dr. JohnDaly (Bethesda, Md.), respectively. Antiarrhythmic drugs were dissolvedin dimethylsulfoxide (DMSO) as stock solutions and stored at 4° C. Finaldrug concentrations were made by serial dilution. The highest DMSOconcentration in bath solution was 0.1% except for BTX, which contained1% DMSO in pipette solution. DMSO at a final concentration of 1% had noeffect on Na⁺ currents. Tetrodotoxin was purchased from EMD Biosciences(San Diego, Calif.) and was dissolved in distilled water as stocksolution at 1 mM. Cells were perfused with an extracellular solutioncontaining (in mM) 65 NaCl, 85 choline-Cl, 2 CaCl₂, and 10 HEPES(titrated with tetramethylammonium-OH to pH 7.4). The pipette(intracellular) solution consisted of (in mM) 100 NaF, 30 NaCl, 10 EGTA,and 10 HEPES (titrated with cesium-OH to pH 7.2).

FIG. 14A shows a family of superimposed inactivation-deficient Na⁺current traces recorded from a cell after 16 passages. The currenttraces were evoked by 8-ms pulses to voltages ranging from −100 to +50mV in 10-mV increments. The inward current evoked by a pulse to −40 mVand the outward current evoked by a pulse to +50 mV are labeled. Underour ionic conditions, these currents were activated around −40 mV andreversed around −10 mV.

For FIG. 14B, conductance was determined from the equationg_(m)=I_(Na)/(E_(m)−E_(Na)), where I_(Na) is the peak current, E_(m) isthe test voltage, and E_(Na) is the estimated reversal potential, andplotted against the corresponding voltage. The plot was fitted with aBoltzmann function. The midpoint voltage (V_(0.5)) is −4.2±0.9 mV. Theholding potential was −140 mV. The conductance curve arises around −40mV and has a slope factor of 15.9±0.7 mV with 50% channels activated at−4.2±0.9 mV (n=7).

We also characterized the steady-state fast inactivation by aconventional two-pulse protocol. Superimposed current traces were evokedby an 8-ms test pulse to +50 mV with 100-ms conditioning pulses,increased in 5-mV increments between −170 to −25 mV. The intervalbetween pulses was 10 seconds (FIG. 14C.). A fraction of Na⁺ currents,˜55%, was non-inactivating even with a conditioning pulse at −25 mV for100 ms (FIG. 14D). Inward Na⁺ currents remained visible during the100-ms conditioning pulses from −40 to −25 mV, before the test pulse wasapplied (FIG. 14C.). These gating phenotypes are similar to thoseobtained by transient transfection of hNav1.4-L443C/A444W Na⁺ channels.

To quantify the relative density of Na⁺ channel expression we measuredboth the peak Na⁺ current amplitude and the cell capacitance ofindividual cells. On average, stably transfected cells expressed 283±58pA/pF (n=8) of peak Na⁺ currents at +50 mV. This level of expression iscomparable to that of rNav1.4 wild-type Na⁺ currents in transientlytransfected cells (e.g., Nau et al., 2003). Thus, impaired fastinactivation alone apparently did not diminish the expression of thesemutant channels in HEK293 cells.

Intact Receptors in hNav1.4-L443C/A444W Inactivation-Deficient MutantChannels for TTX and BTX.

To test whether distinct receptors for TTX and BTX at the externalsurface and within the inner cavity of Na⁺ channels have been altered,we examined the actions of these ligands in cells expressinghNav1.4-L443C/A444W inactivation-deficient mutant channels. Our datashowed that the receptors for TTX and BTX in inactivation-deficient Na⁺channels remained intact (FIGS. 15 and 16) with phenotypes similar totheir wild-type counterparts. An intact BTX receptor suggests that itsbinding site within the inner cavity of mutant Na⁺ channels remainscomparable to that of the wild-type channels [Wang and Wang, 2003],whereas an intact TTX receptor indicates that its binding site at theexternal ion entryway is also comparably normal.

Sensitivity of Inactivation-Deficient Mutant Channels to External BTX

The sensitivity of stably transfected cells expressinginactivation-deficient mutant channels to batrachotoxin (BTX) wasexamined. FIG. 15A demonstrates that BTX prevents the slow decay ofhNav1.4-L443C/A444W mutant Na⁺ currents. A family of superimposedhNav1.4-L443C/A444W Na⁺ current traces was recorded with 800-ms testpulses, ranging from −90 to +50 mV. Initial peak currents were truncatedat this slow time scale. Individual current traces at −40 and +50 mVwere labeled. During depolarization at voltages between −40 mV to +50mV, persistent late Na⁺ currents remained visible even after 800 ms instably transfected HEK293 cells (FIG. 15A). These late Na⁺ currentsdecayed slowly, with a time constant of 258±26 ms at +50 mV (n=5),probably due to an enhanced slow inactivation process [Wang et al.,2003a]. Evidently, hNav1.4-L443C/A444W mutant channels exhibitedmultiple phases of inactivation dependent on the duration ofdepolarization applied (FIGS. 14C and 15A).

Previous experiments have demonstrated that BTX eliminates both fast andslow inactivation of wild-type Nav1.4 Na⁺ channels [Wang and Wang,1998]. In addition, BTX drastically shifts the activation threshold tothe hyperpolarizing direction. We found that inactivation-deficienthNav1.4-L443C/A444W mutant channels remain sensitive to 5 μM BTX appliedwithin the pipette solution. A total of 400 repetitive pulses at +50 mVfor 24 ms were first applied at 1 Hz to facilitate BTX binding. Withoutrepetitive pulses, BTX at 5 μM modified very few inactivation-deficientmutant channels, because BTX did not have access to its receptor whenthe channel was in its closed state. FIG. 15B shows a family ofsuperimposed Na⁺ current traces from −120 to +50 mV at a slow timeframe. Activation of BTX-modified Na⁺ currents occurred at the thresholdof −80 mV (FIG. 15B); once activated, BTX-modified Na⁺ currents weremaintained during 4-s depolarization without evidence of fast or slowinactivation. Because of the leftward shift in activation threshold,large inward Na⁺ currents were recorded between −80 to −20 mV inBTX-treated cells (FIG. 15B), whereas only small inward Na⁺ currentswere evident between −40 to −20 mV in untreated cells (FIG. 15A). Thus,BTX shifts the activation threshold by ˜−40 mV and completely eliminatesthe fast and the slow inactivation of these inactivation-deficienthNav1.4-L443C/A444W mutant channels. These BTX phenotypes are similar tothose found in wild-type Na⁺ channels [Wang and Wang, 1998].

Sensitivity of Inactivation-Deficient Mutant Channels to External TTX

External TTX blocks hNav1.4 wild-type Na⁺ channels with an IC₅₀ of 25 nMin frog oocytes [Chahine et al., 1994]. To determine the TTX affinity ininactivation-deficient hNav1.4-L443C/A444W mutant Na⁺ channels, weconstructed the dose-response curve. FIG. 16A shows the superimposedcurrent traces at various external TTX concentrations ranging from 3 nMto 1 μM. TTX blocked these mutant channels potently, with an IC₅₀ valueof 15.1±0.6 nM (n=5; FIG. 3B) and a Hill coefficient of 1.09±0.04 (n=5)suggesting that, in the hNav1.4-L443C/A444W mutant channel, its externalstructure near the TTX binding site remains similar to that of thewild-type channel. We found no evidence of a time-dependent block byTTX; both the peak and maintained currents were sensitive to TTX blockeven during prolonged depolarization.

Open-Channel Block Induced by Flecainide and Mexiletine DuringDepolarization.

Previous reports indicate that persistent late Na⁺ currents ininactivation-deficient S6 mutant channels are sensitive to block byflecainide and mexiletine in transiently transfected HEK293 cells [Wanget al., 2003b; 2004]. We therefore applied these therapeutic Na⁺ channelblockers to determine whether they also blocked persistent late Na⁺currents in stably transfected HEK293 cells. Persistent late Na⁺currents were indeed far more sensitive to flecainide and mexiletine at10 μM than peak Na⁺ currents. As a result, a strong block of persistentlate Na⁺ currents by these drugs became evident. FIGS. 17A and B showthe blocking characteristics of flecainide and mexiletine, respectively,both at a concentration of 10 μM. Wash-in of these drugs reached theirsteady-state block of late Na⁺ currents within 3-5 min. The open-channelblocking phenotypes shown in FIG. 17 are comparable to those reportedpreviously in inactivation-deficient Na⁺ channels [Wang et al., 2003b,2004]. These results suggest that hNav1.4-L443C/A444W Na⁺ channels instably transfected HEK293 cells interact strongly with flecainide andmexiletine.

By definition, aberrant persistent late Na⁺ currents created bysite-directed mutations, genetic diseases, chemical modifications, orpathological conditions are the results of structural changes of Na⁺channels. Could such alterations instigate an increase or a decrease inreceptor affinities? This possibility would explain two opposing viewson the role of the open-channel block. Unfortunately, directmeasurements of the open-channel block in wild-type Na⁺ channels weredifficult, if not impossible, because these channels displayed a ratherbrief open time [Aldrich et al., 1983]. Putting this uncertainty aside,the pressing clinical challenge is to find therapeutic drugs that cansilence abnormal late Na⁺ currents but leave normal transient Na⁺currents nearly untouched [Wang DW et al., 1997; Maltsev et al., 2001].Inactivation-deficient mutant currents in this cell line may beapplicable as targets to screen such drugs.

The stably transfected cell line of the present invention is used in anassay to screen a large number of compounds for potent open-channelblockers. We determined the blocking phenotype of (S)-propafenone andamiodarone in inactivation-deficient hNav1.4-L443C/A444W mutantchannels. These antiarrhythmic drugs were reported as potentopen-channel blockers either in chemically modifiedinactivation-deficient Na⁺ channels [Benz and Kohlhardt, 1994] or inlate Na⁺ currrents of failing human heart [Maltsev et al., 2001]. FIG.18A shows that (S)-propafenone at 10 μM potently blocks persistent latecurrents; the block reaches its steady state within 3-5 min. The peakNa⁺ currents were also inhibited, but to a lesser extent. In contrast,wash-in of amiodarone at 10 μM was relatively slow (FIG. 18B), unlike(S) propaferone and other drugs shown in FIG. 17 at the sameconcentration. Most, if not all, persistent late currents were inhibitedby 10 μM amiodarone over a period of 15 min. The peak currents were alsoreduced slowly, but again to a lesser extent. Wash-out of amiodaroneblock was slow and incomplete over a period of ≧30 min. Together, theseresults strongly suggest that this cell line expressinghNav1.4-L443C/A444W mutant channels can be used to identify potentopen-channel blockers. The fact that at their therapeutic plasmaconcentration antiarrhythmic mexiletine (2.8-11.2 μM; Roden, 2001),flecainide (0.5-2.4 μM), amiodarone (0.77-3.1 μM), and propafenone(0.5-1.5 μM); [Steurer et al., 1991] effectively block persistentcurrents in this cell line supports such an assessment. We found thatantiarrhythmic flecainide, mexiletine, propaferone, and amiodarone,potently blocked persistent late Na⁺ currents of inactivation-deficientmutant channels (FIG. 17 and 18). Previous investigations give twoconflicting hypotheses regarding the role of the open-channel block. Onthe one hand, a quaternary ammonium derivative of lidocaine, QX-314,does not block the inactivation-deficient Na⁺ currents inpronase-treated Na⁺ channels [Cahalan, 1978; Yeh, 1978]. In IFM/QQQmutant channels, the open-channel affinity for lidocaine or cocaine is3- to 10-fold less than the inactivated-channel affinity [Bennett etal., 1995b; O'Leary and Chahine, 2002]. These results all lead to ahypothesis that the inactivated state plays a dominant role inQX-314/lidocaine/cocaine block of Na⁺ channels, consistent with themodulated receptor hypothesis [Hille, 1977; Hondeghem and Katzung, 1977;Bean et al., 1983]. Results from a weak open-channel block in IFM/QQQmutant channels by flecainide, RAD-243, and disopyramide [Grant et al.,2000] gave additional support for this hypothesis. On the other hand,local anesthetics and antiarrhythmic drugs potently block persistentlate Na⁺ currents that were generated under different conditions. Amongthese conditions are (1) chloramine-T-treated squid axons [Wang et al.,1987], (2) mutations that cause genetic diseases [Wang DW et al., 1997;1999; Nagatomo et al., 2000], (3) hypoxia/failing cardiac myocytes [Juet al., 1992; 1996; Maltsev et al., 2001], or (4) inactivation-deficientS6 mutants [Wang et al., 2003b; 2004]. These results indicate that theopen state of the Na⁺ channel binds strongly with lidocaine, QX-314,mexiletine, and flecainide and lead to an opposite hypothesis that theopen-channel block plays an important therapeutic role in vivo.

1. A method for assessing the potential of a compound to function as an anti-arrhythmic agent comprising: (a) providing a cell that expresses a recombinant mutant Nav1 sodium channel protein; (b) measuring a first plateau current in said cell; (c) exposing said cell to a test compound; (d) measuring a second plateau current in said cell; and (e) comparing said first and second currents whereby a lower second current indicates that said test compound is a potential anti-arrhythmic agent; said mutant sodium channel protein having an amino acid sequence in which one or more amino acids among the ten amino acids occurring at the carboxy end of the S6 segments of D1, D2, D3 or D4 domains of a mammalian Nav1 protein differs from the amino acid in wild-type Nav1 by substitution with tryptophan, phenylalanine, tyrosine or cysteine.
 2. The method of claim 1 wherein said mammalian Nav 1 protein is selected from Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, or Nav 1.8.
 3. The method of claim 2 wherein said mammalian Nav 1 protein is Nav 1.4 or Nav 1.5.
 4. A method for assessing the potential of a compound as an anti-arrhythmic agent comprising: (a) providing a cell that expresses a recombinant mutant Nav1 sodium channel protein; (b) measuring a first plateau current in said cell; (c) exposing said cell to a test compound; (d) measuring a second plateau current in said cell; and (e) comparing said first and second currents whereby a lower second current indicates that said test compound is a potential anti-arrhythmic agent; said mutant sodium channel protein having an amino acid sequence in which at least one amino acid chosen from amino acids 19, 21 and 22 of the S6 segment of D1 and amino acids 23 and 24 of the S6 segment of the D4 domain of a mammalian Nav1 protein differs from the amino acid in wild-type Nav1 by substitution with tryptophan, phenylalanine, tyrosine or cysteine.
 5. The method of claim 4 wherein said mammalian Nav 1 protein is selected from Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, or Nav 1.8.
 6. The method of claim 5 wherein said mammalian Nav 1 protein is Nav 1.4 or Nav 1.5.
 7. A method for assessing the potential of a compound as an anti-arrhythmic agent comprising: (a) providing a cell that expresses a recombinant mutant Nav1.4 or Nav1.5 sodium channel protein; (b) measuring a first plateau current in said cell; (c) exposing said cell to a test compound; (d) measuring a second plateau current in said cell; and (e) comparing said first and second currents whereby a lower second current indicates that said test compound is a potential anti-arrhythmic agent; said mutant sodium channel protein having an amino acid sequence in which at least one amino acid chosen from amino acids L435, L437, A438, I1589 and I1590 of wild-type rNav1.4 is replaced by tryptophan, phenylalanine or tyrosine, or in the case of L437 additionally with cysteine.
 8. A method according to claim 1 wherein said cell is chosen from a human embryonic kidney cell and a Chinese hamster ovary cell.
 9. A method according to claim 1 wherein one or more wild-type amino acids are replaced with tryptophan.
 10. A method according to claim 3 wherein the mammalian Nav1.4 or Nav1.5 is rat or human Nav1.4 or Nav1.5 and a leucine corresponding to L437 of rNav1.4 is replaced with cysteine.
 11. A method according to claim 10 wherein L437 is replaced with cysteine and one or both of a leucine and an alanine corresponding to L435 and A438 respectively of rNav1.4 are replaced with tryptophan.
 12. The method according to claim 3 wherein the mammalian Nav1.4 or Nav1.5 is rat or human Nav1.4 or Nav1.5.
 13. The method according to claim 12 wherein an alanine corresponding to A438 and an isoleucine corresponding to I1589 in rNav1.4 are replaced.
 14. The method according to claim 13 wherein said alanine and isoleucine are replaced by tryptophan.
 15. A cell comprising a nucleic acid that encodes a recombinant mutant mammalian Nav1 protein, said mutant protein having a sequence in which one or more amino acids among the ten amino acids occurring at the carboxy end of the S6 segments of D1, D2, D3 or D4 domains of mammalian Nav1 differs from the amino acid in wild-type Nav1 by substitution with tryptophan, phenylalanine, tyrosine or cysteine.
 16. The cell of claim 15 wherein said mammalian Nav 1 protein is selected from Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, Nav 1.8, or Nav 1.9.
 17. The cell of claim 16 wherein said mammalian Nav 1 protein is Nav 1.4 or Nav 1.5.
 18. A cell comprising a nucleic acid that encodes a recombinant mutant mammalian Nav1 protein, said mutant protein having an amino acid sequence in which at least one amino acid chosen from amino acids 19, 21 and 22 of the S6 segment of D1 and amino acids 23 and 24 of the S6 segment of the D4 domain of mammalian Nav1 differs from the amino acid in wild-type Nav1 by substitution with tryptophan, phenylalanine, tyrosine or cysteine.
 19. The cell of claim 18 wherein said mammalian Nav 1 protein is selected from Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, Nav 1.8, or Nav 1.9.
 20. The cell of claim 19 wherein said mammalian Nav 1 protein is Nav 1.4 or Nav 1.5.
 21. A human cell comprising a nucleic acid that encodes a mutant mammalian Nav1.4 or Nav1.5 protein, said mutant sodium channel protein having an amino acid sequence in which at least one amino acid chosen from amino acids L435, L437, A438, I1589 and I1590 of wild-type rat Nav1.4 is replaced by tryptophan, phenylalanine or tyrosine, or in the case of L437 additionally with cysteine.
 22. A cell according to claim 15 wherein said mutant sodium channel protein gives rise to sodium channels exhibiting plateau currents of greater than 1 nanoamp.
 23. A screen for assessing the potential of a compound to treat a pathological condition manifested by an increased late sodium current in a heart comprising: (a) providing a cell that expresses a recombinant mutant Nav1 sodium channel protein; (b) measuring a first plateau current in said cell; (c) exposing said cell to a test compound; (d) measuring a second plateau current in said cell; and (e) comparing said first and second currents whereby a lower second current indicates that said test compound is a potential anti-arrhythmic agent; said mutant sodium channel protein having an amino acid sequence in which one or more amino acids among the ten amino acids occurring at the carboxy end of the S6 segments of D1, D2, D3 or D4 domains of mammalian Nav1 differs from the amino acid in wild-type Nav1 by substitution with tryptophan, phenylalanine, tyrosine or cysteine.
 24. The method of claim 23 wherein said mammalian Nav1 protein is selected from Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, or Nav 1.8.
 25. The method of claim 24 wherein said mammalian Nav 1 protein is Nav 1.4 or Nav 1.5.
 26. A screen for assessing the potential of a compound to treat a pathological condition manifested by an increased late sodium current in a heart comprising: (a) providing a cell that expresses a mutant Nav1 sodium channel protein; (b) measuring a first plateau current in said cell; (c) exposing said cell to a test compound; (d) measuring a second plateau current in said cell; and (e) comparing said first and second currents whereby a lower second current indicates that said test compound is a potential anti-arrhythmic agent; said mutant sodium channel protein having an amino acid sequence in which at least one amino acid chosen from amino acids 19, 21 and 22 of the S6 segment of D1 and amino acids 23 and 24 of the S6 segment of the D4 domain of mammalian Nav1.4 or Nav1.5 differs from the amino acid in wild-type Nav1 by substitution with tryptophan, phenylalanine, tyrosine or cysteine.
 27. The method of claim 26 wherein said mammalian Nav1 protein is selected from Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, or Nav 1.8.
 28. The method of claim 27 wherein said mammalian Nav 1 protein is Nav 1.4 or Nav 1.5.
 29. A screen for assessing the potential of a compound to treat a pathological condition manifested by an increased late sodium current in a heart comprising: (a) providing a cell that produces mutant Nav1.4 or Nav1.5 sodium channel protein; (b) measuring a first plateau current in said cell; (c) exposing said cell to a test compound; (d) measuring a second plateau current in said cell; and (e) comparing said first and second currents whereby a lower second current indicates that said test compound is a potential anti-arrhythmic agent; said mutant sodium channel protein having an amino acid sequence in which at least one amino acid chosen from amino acids L435, L437, A438, I1589 and I1590 of wild-type rNav1.4 is replaced by tryptophan, phenylalanine or tyrosine, or in the case of L437 additionally with cysteine.
 30. A screen according to claim 26 wherein said cell is chosen from a human embryonic kidney cell and a Chinese hamster ovary cell.
 31. A screen according to claim 26 wherein one or more wild-type amino acids are replaced with tryptophan.
 32. A screen according to claim 26 wherein the mammalian Nav1.4 or Nav1.5 is rat or human Nav1.4 or Nav1.5 and a leucine corresponding to L437 of rNav1.4 is replaced with cysteine.
 33. A screen according to claim 32 wherein L437 is replaced with cysteine and one or both of a leucine and an alanine corresponding to L435 and A438 respectively of rNav1.4 are replaced with tryptophan.
 34. A screen according to claim 26 wherein the mammalian Nav1.4 or Nav1.5 is rat or human Nav1.4 or Nav1.5.
 35. A screen according to claim 34 wherein an alanine corresponding to A438 and an isoleucine corresponding to I1589 in rNav1.4 are replaced.
 36. A screen according to claim 35 wherein said alanine and isoleucine are replaced by tryptophan.
 37. A screen according to claim 26 wherein said mutant sodium channel protein gives rise to sodium channels exhibiting plateau currents of greater than 1 nanoamp. 